Mammals and invertebrates possess endogenous circadian pacemakers (clocks) within their nervous systems that regulate the expression of daily rhythms in various physiological processes including human sleep/wake states and mental performance. In the fruitfly Drosophila, a molecular genetic approach has been applied to identify and characterize the genes that determine the properties of circadian rhythms. This proposal will focus on the molecular analysis of a Drosophila gene known as miniature-dusky (m- dy). Our previous genetic analyses have shown that certain dy mutations lengthen the period of two types of circadian rhythms. Interestingly, these and other dy alleles also reduce cell size in the adult wing. Using molecular genetic techniques, we have cloned and defined the physical limits of the m-dy locus. Transcriptional activity of the locus has been assessed using RNA blot and RNase protection procedures. The latter studies have identified a transcript that is expressed in the Drosophila head, the anatomical site of the circadian pacemaker. This transcript cannot be detected in head RNA from dyn3, a mutant which has altered circadian rhythms. Moreover, the developmental expression of a different transcript is altered by a mutation known as dy73. The present application proposes molecular studies to understand the cellular function of the m-dy locus. The complete developmental pattern of gene expression will be delineated in wild-type flies and several m-dy mutants including dyn3 and dy73. P-mediated DNA transformation techniques will be used to study the relationship between the wing and circadian rhythm phenotypes of dy mutants. Sequence analyses of wild-type and mutant DNAs will be carried out to deduce the primary structure of and define functional domains within m-dy proteins. Antisera will be generated and used in immunolocalization studies to examine the pattern of protein expression within the Drosophila nervous system and other tissues. Information about the structure and distribution of this protein is essential for determining how its cellular function is related to the expression of circadian periodicity. An understanding of the cellular and molecular bases of circadian regulation in any organism has important implications for the etiology of certain human psychiatric and sleep disorders.